Camptothecin analogs as potent inhibitors of topoisomerase I

ABSTRACT

A method for synthesizing camptothecin and camptothecin analogs using a novel hydroxyl-containing tricyclic intermediate and the camptothecin analogs produced by the process. The camptothecin analogs are effective inhibitors of topoisomerase I and show anti-leukemic and anti-tumor activity.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 07/032,449 filed Mar. 31, 1987, now U.S. Pat. No.4,894,456.

BACKGROUND OF THE INVENTION

1. Field of the Invention:

The invention relates to camptothecin and analogs thereof which showlife prologation effects in various leukemia systems such as P-388 andL-1210; inhibition of animal tumors such as B-16 melanoma and are potentinhibitors of topoisomerase I and a method of synthesizing the same bymeans of a novel hydroxyl-containing tricyclic intermediate. Theinvention also relates to a method for inhibiting topoisomerase I andtreating colorectal cancer.

2. Discussion of the Background:

Camptothecin is a pentacyclic alkaloid initially isolated from the woodand bark of Camptotheca acuminata by Wall et al (M. E. Wall, M. C. Wani,C. E. Cook, K. H. Palmer, A. T. McPhail, and G. A. Sim, J. Am. Chem.Soc., 94, 388 (1966).

Camptothecin is highly biologically active and displays stronginhibitory activity toward the biosynthesis of nucleic acids.Additionally, camptothecin exhibits potent anti-tumor activity againstexperimentally transplanted carcinoma such as leukemia L-1210 in mice orWalker 256 tumor in rats

Several methods for the synthesis of camptothecin and camptothecinanalogs are known. These synthetic methods include (i) methods in whichnaturally occurring camptothecin is synthetically modified to produce anumber of analogs and (ii) totally synthetic methods.

U.S. Pat. Nos. 4,604,463; 4,545,880; and 4,473,692 as well as EuropeanPatent Application 0074256 are examples of the former type of syntheticstrategy. Additional examples of this strategy can be found in JapanesePatents 84/46,284; 84/51,287; and 82/116,015. These methods requirenaturally occurring camptothecin which is difficult to isolate and hencethese methods are not suitable for the production of large quantities ofcamptothecin or analogs.

Examples of a variety of totally synthetic routes to camptothecin andcamptothecin analogs can be found in the following references: Sci. Sin.(Engl. Ed),. 21(1), 87-98 (1978); Fitoterpapia, 45(3), 87-101 (1974);Yakugaku Zashi, 92(6), 743-6 (1972); J. Org. Chem., 40(14), 2140-1(1975); Hua Hsueh Hsueh Pao, 39(2), 171-8 (1981); J. Chem. Soc., PerkinTrans 1, (5), 1563-8 (1981); Heterocycles, 14(7), 951-3 (1980); J. Amer.Chem. Soc., 94(10), 3631-2 (1972); J. Chem. Soc. D, (7), 404 (1970) andU.S. Pat. No. 4,031,098.

Wani et al, J. Med. Chem., 23, 554 (1980) discloses a synthesis ofcamptothecin and camptothecin analogs which involves the reaction of atricyclic compound with a suitably substituted orthoaminoaldehyde toyield desoxycamptothecin as shown in Equation 1 below. ##STR1##

Desoxycamptothecin is then treated with oxygen to give camptothecinanalogs. A major disadvantage of this procedure is the insolubility ofdesoxycamptothecin and its analogs, requiring large solvent volumes inthe final step. A poor yield of the oxygenation product results underthese conditions.

There exists a need, therefore, for a high-yield, efficient synthesis ofcamptothecin and camptothecin analogs which does not require priorisolation of naturally occurring camptothecin.

A need also exists for a method of synthesizing camptothecin andcamptothecin analogs which does not suffer from insolubility problems ofintermediate compounds and the resulting low yields.

A further need exists for new camptothecin analogs which can besynthesized in an efficient, high-yield manner and which show goodbiological activity.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a methodof synthesizing camptothecin and camptothecin analogs in high yield in atotally synthetic process.

Another object of the present invention is to provide a process forsynthesizing camptothecin and camptothecin analogs which does not sufferfrom problems associated with the insolubility of intermediatecompounds.

A further object of the invention is to provide a process for thepreparation of camptothecin and camptothecin analogs which can be easilymodified to produce a variety of analog structures.

Still a further object of the present invention is to providecamptothecin analogs which show good antitumor activity and otherdesirable biological activities.

A further object of the present invention is to provide a method fortreating colorectal cancer by administering an antitumor or anticanceramount of a camptothecin compound.

These objects and other objects of the present invention which willbecome apparent from the following specificiation have been achieved bythe present method for the synthesis of camptothecin and camptothecinanalogs, which includes the steps of:

cyclizing a compound of the formula shown below, wherein X is an organicgroup which is converted to a carbonyl group when treated with an acid,##STR2## to form a lactone having the formula ##STR3## deprotecting saidlactone to form a hydroxylcontaining tricyclic compound having theformula shown below, and ##STR4## reacting said hydroxyl-containingtricyclic compound with a substituted ortho-amino compound of theformula ##STR5## wherein n=1-2 and wherein each R is selected from thegroup consisting of cyano, methylenedioxy, formyl, hydroxy, C₁₋₈ alkoxy,nitro, amino, chloro, bromo, iodo, fluoro, C₁₋₈ alkyl, trifluoromethyl,aminomethyl, azido, amido and hydrazino groups; R² is H₁ or C₁₋₈ alkyl;and R³ is the side-chain of any of the twenty naturally occurring aminoacids.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A(a), 1A(b) and FIG. 1B illustrates the synthesis of thehydroxyl-containing tricyclic compound 11, according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The camptothecin and camptothecin analogs produced by the process of thepresent invention are racemic, and therefore contain both the(R)-20-hydroxy and (S)-20-hydroxy camptothecin compounds. Naturallyoccurring camptothecin belongs to the 20(S) series of compounds.Therefore, the compounds produced by the process of the presentinvention contain a mixture of the natural and non-naturally occurringcompounds.

The camptothecin analogs of the present invention have the basiccamptothecin structural framework shown below in which the A ring issubstituted. ##STR6##

Substituents within the scope of the present invention include hydroxy,nitro, amino, chloro, bromo, iodo, fluoro, C₁₋₈ alkyl, C₁₋₈ alkoxy,trifluoromethyl, aminomethyl, amido, hydrazino, azido, formyl, and cyanogroups as well as groups comprising amino acids bonded to the aromaticring via the amino-nitrogen atom. Preferred alkyl groups include methyl,ethyl, propyl, butyl, isopropyl, isobutyl and sec-butyl groups.Preferred alkoxy groups include methoxy, ethoxy, propoxy and isopropoxygroups.

The preferred amino acid groups are the 20 naturally occuring aminoacids having an (L) configuration. These amino acids are well known tothose skilled in the art.

Additionally, two substituents on the A ring may be joined together toform a bifunctional substituent such as the methylenedioxy group.Methylenedioxy substituents may be bonded to any two consecutivepositions in the A ring, for example, the 9,10; 10,11 or 11,12positions.

Preferred substituents include the hydroxy, amino, cyano andmethylenedioxy substituents. A particularly preferred substituent is themethylenedioxy group.

Particularly preferred compounds within the scope of the inventioninclude 11-methoxy-20(RS)-camptothecin, 11-hydroxy-20(RS)-camptothecin,10-hydroxy-20(RS)-camptothecin, 9-methoxy-20(RS)-camptothecin,9-hydroxy-20(RS)-camptothecin, 10-nitro-20(RS)-camptothecin,10-amino-20(RS)-camptothecin, 9-nitro-20(RS)-camptothecin,9-amino-20(RS)-camptothecin, 11-nitro-20(RS)-camptothecin,11-amino-20(RS)-camptothecin, 10,11-dihydroxy-20(RS)-camptothecin,10-chloro-20(RS)-camptothecin, 10-methyl-20(RS)-camptothecin,11-formyl-20(RS)-camptothecin and 11-cyano-20(RS)-camptothecin and10,11-methylenedioxy-20(RS)-camptothecin.

Also included within the scope of the present invention are compounds inwhich the A ring of the camptothecin structure is modified to contain ahetero atom. The modified structures can have an A ring which contains 5or 6 atoms and the hetero atom may be a nitrogen, sulfur or oxygen atom.These compounds may be represented by the general structure shown belowin which the A ring is an aromatic 5 or 6 membered ring containing thehetero atom X. ##STR7##

Preferred compounds having a modified A ring structure include compoundsin which the A ring is a 6 membered nitrogen-containing aromatic ringand compounds in which the A ring is a 5 membered sulfur-containingaromatic ring. Particularly preferred compounds are10-aza-20(RS)-camptothecin and A-nor-9-thia-20(RS)-camptothecin.

The camptothecin analogs noted above may be synthesized according to themethod of the present invention by reacting a tricyclic compoundcontaining a 20-hydroxyl group with an appropriately substitutedortho-amino aromatic aldehyde or ketone. Camptothecin analogs having analkyl substituent on C₇ are produced when the appropriate ortho-aminoketone is used.

An important step in the method of the present invention is thesynthesis of the hydroxyl-containing tricyclic compound having theformula I shown below and in which R is a hydroxyl group. ##STR8##

(I)

A synthetic method previously developed by the present inventors (J.Med. Chem., 23, 554 (1980)) utilized a related but structurallydifferent tricyclic compound (formula I, R=H). In that method, thetricyclic compound was reacted with a suitable orthoaminoaldehyde underalkaline or acidic conditions to yield a desoxycamptothecin. Thedesoxycamptothecin was then reacted with oxygen to give camptothecinanalogs in which R is OH. A major disadvantage of this procedure is theinsolubility of the desoxycamptothecin and its analogs, requiring largesolvent volumes in the final step and giving poor yields of theoxygenation product.

In contrast, the method of the present invention synthesizes the keytricyclic intermediate (11) according to FIG. 1. The synthesis ofcompounds 1-9 was disclosed in Wani et al, J. Med. Chem., 23, 554(1980). In further contrast to the previous synthesis, the presentmethod introduces the 20-hydroxyl group earlier in the syntheticsequence and then forms the lactone ring to give compound 10. Afterdeprotection of the carbonyl group, the key hydroxyl-containingtricyclic compound 11 is obtained.

The protection of the carbonyl group in compound 3 can be performedusing any appropriate organic protecting group which can be removed orconverted into a carbonyl group upon treatment with acid. The carbonylgroup is thereby "deprotected". These protecting groups are well knownto those familiar with synthetic chemistry, and include acetals, ketals,thioacetals, thioketals, etc. Preferred protecting groups have 2-6carbon atoms. An especially preferred protecting group is --OCH₂ CH₂O--.

As a consequence of prior introduction of the hydroxyl group into thetricyclic compound 11, the desired pentacyclic analogs are reduced inone step by reaction with the appropriate ortho-amino carbonylcompounds. Both compound 11 and the corresponding ketonic synthons arevery soluble in organic solvents whereas the pentacyclic product isinsoluble. Hence, the oxygenation step, i.e, the introduction of thehydroxyl group, is conveniently carried out at the tricyclic stagerather than on the insoluble pentacyclic desoxy analogs.

Tricyclic compound 11 is then reacted with a suitably substitutedortho-amino aldehyde or ketone to give a camptothecin analog.Substituted ortho-amino aldehydes and ketones within the scope of thepresent invention include ortho-amino aldehydes and ketones having atleast one additional substituent on the aromatic ring. This substituentmay be at one or more of the positions equivalent to the 9, 10, 11 or 12positions of the A ring of the final camptothecin structure as shownbelow. ##STR9##

Preferred substituted ortho-amino aldehydes and ketones havesubstituents in one or more of the equivalent 9, 10, or 11 positions.

The substituents on the substituted ortho-aminoaldehyde or ketoneinclude hydroxy, nitro, amino, C₁₋₈ alkyl, chloro, bromo, iodo, fluoro,methylenedioxy (--O--CH₂ --O--), C₁₋₈ alkoxy, trifluoromethyl,aminomethyl, amido, hydrazino, azido, formyl, and cyano groups as wellas groups comprising amino acids bonded to the aromatic ring through theamino-nitrogen atom. Preferred examples include the hydroxy, amino,cyano and methylenedioxy substituents. A particularly preferredsubstituent is the methylenedioxy group.

When an ortho-amino ketone is reacted with tricyclic compound 11, acamptothecin analog having an alkyl substituent at C₇ is produced.Preferred orthoamino ketones are those in which R² is an alkyl grouphaving 1-8 carbon atoms. Especially preferred orthoamino ketones areortho-aminoacetophenone and orthoaminopropiophenone.

The ortho-amino aldehydes and ketones may be substituted by a grouphaving the formula

    --NH--CHR.sup.3 --COOH

wherein R³ is a side-chain of one of the twenty naturally occurringamino acids. The amino acid substituent is bonded to the aromatic ringvia the nitrogen atom and may be bonded to any position on the aromaticring equivalent to the 9, 10, 11 or 12 positions of the A ring of thefinal camptothecin structure.

The ortho-amino aldehydes and ketones may be in the free carbonyl formor in a form in which the carbonyl of the aldehyde or ketone isprotected by a standard protecting group. These protecting groups arewell known to those skilled in the art. Ortho-amino aldehydes andketones in the free carbonyl form and in the protected carbonyl form areconsidered within the scope of the present invention and are suitablefor use in the present method.

The reaction in which the hydroxyl group is introduced into thetricyclic intermediate compound, i.e, the cyclizing step, can beeffected by any suitable reaction which will introduce the hydroxylgroup at the appropriate position of compound 9 without causingsignificant side reactions such as degradation of compound 9 itself.

The reaction is preferably conducted in the presence of a basiccatalyst. Suitable basic catalysts include both inorganic and organicbases. Preferred inorganic bases include, for example, sodium andpotassium carbonate and sodium and potassium bicarbonate. Preferredorganic bases include hindered bases such as triethylamine anddiisopropylamine. A particularly preferred basic catalyst is potassiumcarbonate.

The reaction in which the hydroxyl group is introduced can be performedin the presence of any polar or non-polar solvent in which the reactantsare suitably soluble to react. Preferred are polar organic solvents suchas methanol, ethanol, propanol, butanol and dimethylformamide. Ethersolvents, including crown ethers may also be used.

The oxygen of the hydroxyl group is generally derived from molecularoxygen which is bubbled through the reaction solution. Although the useof oxygen is preferred, other sources of oxygen, such as air, may alsobe used. Other oxidizing agents such as hydrogen peroxide, leadtetraacetate and selenium dioxide may also be employed.

This reaction is preferably conducted at room temperature although thespecific reaction temperature will be dependent on the specific reactionconditions and reactants used.

The deprotection of the carbonyl group in compound 10 is accomplished bytreatment with acid. Suitable acids include mineral acids such as HC1,H₂ SO₄, HNO₃, and H₃ PO₄, as well as organic acids such as alkanoicacids having 1-10 carbon atoms, proferably acetic acid, and C₁₋₁₂arylsulfonic acids, especially p-toluenesulfonic acid. The deprotectionof a carbonyl group in this manner is well known to those skilled in theart.

The tricyclic compound 11 is then reacted with a substituted ortho-aminoaldehyde or ketone in the presence of an acid or base catalyst. The basecatalyst is preferably any of the base catalysts noted above incyclizing compound 9 to form compund 10, i.e., for the introduction ofthe hydroxyl group into tricyclic compound 11. The acid catalyst ispreferably a mineral acid such as for example HC1, H₂ SO₄, HNO₃, and H₃PO₄, or organic acids such as C₁₋₈ alkanoic acids and C₁₋₁₂ arylsulfonicacids, especially p-toluenesulfonic acid.

The reaction of compound 11 with an appropriate ortho-amino compound maybe carried out neat or in the presence of a polar or non-polar solvent.Preferred polar solvents are the C₁₋₆ alcohols, ethers anddimethylformamide. Preferred non-polar solvents are branched or straightchained alkyl hydrocarbons having 4-10 carbon atoms and aromatichydrocarbons having 6-20 carbon atoms. An especially preferred solventis toluene.

The reaction of the hydroxyl-containing tricyclic compound with theoptionally substituted ortho-amino compound is generally conducted withheating at reflux. Reaction times will vary depending on the particularreactants but are generally in the range from about 10 minutes to 24hours. Preferred reaction times are in the range of 2-10 hours.

Also within the scope of the present invention are camptothecin analogsprepared by hydrolyzing the lactone ring of the camptothecin analogsprepared as described above using an aqueous alkali solution to preparethe alkali metal salts of camptothecin analogs in which the lactone ringhas been opened to form the corresponding hydroxyl and carboxylatefunctional groups. The hydrolysis is typically carried out in aqueoussolution using one equivalent of an alkali metal hydroxide. Preferredalkali metal hydroxides are potassium hydroxide and sodium hydroxide,with sodium hydroxide being particularly preferred.

The hydrolysis reaction may be conducted at any temperature which allowsadequate reaction of the camptothecin analog and the aqueous alkalimetal hydroxide so long as the temperature is sufficiently low toprevent decomposition of the starting materials. Suitable temperaturesare from about 5°-50° C. with preferred temperatures being approximatelyroom temperature. After hydrolysis, the metal salt of the camptothecinanalog may be isolated and purified by standard recrystallization orchromatographic processes. A particularly preferred salt is the sodiumsalt of 10,11-methylenedioxy-20(RS)-camptothecin.

The camptothecin analogs of the present invention have excellentbiological activity. As used herein, "biological activity" refers to theability of the camptothecin analogs to inhibit topoisomerase enzymes, inparticular topoisomerase I, and their ability to exert anti-leukemicactivity. Anti-leukemic activity may be determined by the ability of therespective compounds to inhibit L-1210 mouse leukemia cells. Althoughanti-leukemic activity is demonstrated here by the activity of theparticular compounds against L-1210 mouse leukemia cells, other knownanti-leukemic and anti-tumor in vitro and in vivo models may be used aswell to determine anti-leukemic activity.

Camptothecin analogs, in particular 9-amino-, 10-amino-,10,11-methylenedioxy- and the sodium salt of10,11-methylenedioxy-camptothecin are highly active as topoisomerase Iinhibitors as shown in in vitro assays and also in in vivo tumor modelssuch as human colorectal xenograft tumor lines. These camptothecinanalogs have been shown to cause remission of tumors in human colorectalxenograft lines such as human colon cancer HT-29, colon cancer CASE andSW 48 tumors. (See B. C. Giovanella, J. S. Stehlin, M. E. Wall, M. C.Wani, A. W. Nicholas, L. F. Liu, R. Silber and M. Potmesil, HighlyEffective DNA Topoisomerase-I Targeted Chemotherapy of Human ColonCancer in Xenografts, Science, 246:1989, P.1046. In addition, certainhuman liver and lung xenographs are strongly inhibited by these analogs.

The camptothecin analogs of the present invention, and in particular9-amino-, 10-amino-, 10,11-methylenedioxyand the sodium salt of10,11-methylenedioxycamptothecin, therefore, provide a method oftreating colorectal cancer by administering to a mammal, and inparticular a human patient, an anti-tumor effective amount of thecamptothecin analog. The high antitopoisomerase I activity of thesecamptothecin analogs will allow the treatment of colon and rectaladenocarcinomas in mammals. Similarly, these compounds may be useful forliver and lung neoplasms.

The mouse anti-leukemic activity of the various ring A oxygenatedcamptothecin analog is shown in Table I. Similar data for nitrogenanalogs and for ring A modified analogs are shown in Tables II and III,respectively. In most cases camptothecin or an analog with well-definedactivity was also assayed at the same time as a positive control, andthe data are shown in the table footnotes. In this manner the relativeantileukemic activity of the various compounds can be compared. Thebiological activity of additional camptothecin analogs is described inJ. Med. Chem., 23, pages 554-560 (1980).

The ability of camptothecin to inhibit topoisomerase I has been shown.See J. Biol. Chem., 260, 14873-73 (1985).

                                      TABLE I                                     __________________________________________________________________________    Comparative Activities and Potencies of Ring A                                Oxygenated Camptothecin Analogues in Mouse Leukemia Assays.sup.a,b            Camptothecin                                                                              max % T/C                                                                            no. cures                                                                          K.sub.E.sup.c at max                                                                active dose                                                                          toxic dose,                              derivative  (dose, mg/kg)                                                                        out of 6                                                                           % T/C range, mg/kg                                                                         mg/kg                                    __________________________________________________________________________    10-OH-20(S).sup.a,d                                                                       297 (3.1)                                                                            0          0.4.sup.e -3.1                                                                       6.25                                     10-OMe-20(S).sup.a,d                                                                      167 (1.6)                                                                            0          0.4.sup.e -1.6                                                                       3.1                                      11-OH-20(RS).sup.b,f                                                                      357 (60.0)                                                                           3    J 5.68                                                                              7.5.sup.e -60.0.sup.g                                                                >60.0                                    10,11-diOMe-20(RS).sup.b,h                                                                inactive                 >50.0.sup.g                              10,11-OCH.sub.2 O-20(RS).sup.b,i                                                          325 (2.0)                                                                            2    J 5.97                                                                              2.0.sup.e -4.0                                                                       >8.0                                     10-OCH.sub.2 CO.sub.2 Na-20(S).sup.a,d                                                    inactive                                                          10-Et.sub.2 N(CH.sub.2).sub.2 O-20(S).sup.a,d                                             183 (16.0)                                                                           0          2.0.sup.e -32.0.sup.g                                                                >32.0                                    __________________________________________________________________________     .sup.a Denotes testing in P388 system; treatment schedule Q04DX03; % T/C      survival time of treated/control animals × 100; IP using Klucel         emulsifier.                                                                   .sup.b Denotes testing in L1210 system; treatment schedule Q04DX02; % T/C     = survival time of treated/control animals × 100; IP using Klucel       emulsifier.                                                                   .sup.c Log.sub.10 of initial tumor cell population minus log.sub.10 of        tumor cell population at end of treatment.                                    .sup.d 20(S)Camptothecin and 20(S)camptothecin sodium were used as            reference standards; for 20(S)camptothecin, % T/C (4.0 mg/kg) = 197; for      40(S)camptothecin sodium, % T/C (40.0 mg/kg) = 212.                           .sup.e Lowest dose administered.                                              .sup.f 20(S)camptothecin and 20(S)camptothecin sodium were used as            reference standards: for 20(S)camptothecin, % T/C (8.0 mg/kg) = 164; for      20(S)camptothecin sodium, % T/C (40.0 mg/kg) = 178.                           .sup.g Highest dose administered.                                             .sup.h 20(S)camptothecin sodium was used as a reference standard: % T/C       (25.0 mg/kg) = 206.                                                           .sup.i 20(S)camptothecin was used as a reference standard: % T/C (5 mg/kg     = 166.                                                                   

                                      TABLE II                                    __________________________________________________________________________    Comparison of Activities and Potencies of Ring A Nitrogen Substituted         and                                                                           Ring A Nitrogen/Oxygen Disubstituted Analogues in L-1210 Mouse Leukemia       Assays.sup.a                                                                  Camptothecin                                                                             max % T/C                                                                            no. cures                                                                          K.sub.E.sup.b at max                                                                active dose                                                                          toxic dose,                               derivative (dose, mg/kg)                                                                        out of 6                                                                           % T/C range, mg/kg                                                                         mg/kg                                     __________________________________________________________________________    10-NO.sub.2 -20(RS).sup.c                                                                219 (15.5)                                                                           1    J 5.86                                                                              7.5.sup.d -15.5                                                                      31.0                                      10-NH.sub.2 -20(RS).sup.c                                                                329 (8.0)                                                                            3    J 5.86                                                                              4.0.sup.d -16.0                                                                      32.0                                      10-NHAc-20(RS).sup.c                                                                     318 (40.0)                                                                           1    J 5.86                                                                              5.0.sup.d -40.0.sup.e                                                                >40.0                                     9-NO.sub.2 -20(S).sup.f                                                                  348 (10.0)                                                                           5    J 5.86                                                                              2.5.sup.d -20.0                                                                      40.0.sup.e                                12-NO.sub.2 -20(S).sup.f                                                                 151 (40.0)                                                                           0    0.34  2.5.sup.d -40.0.sup.e                                                                >40.0                                     9-NH.sub.2 -20(S).sup.f                                                                  348 (2.5)                                                                            4    J 5.86                                                                              2.5.sup.d -5.0                                                                       10.0                                      12-NH.sub.2 -20(S).sup.f                                                                 inactive                                                           9-NO.sub.2 -10-OMe-20(S).sup.f                                                           160 (40.0)                                                                           0    1.03  2.5.sup.d -40.0.sup.e                                                                >40.0                                     9-NH.sub.2 -10-OMe-20(S).sup.f                                                           186 (40.0)                                                                           0    2.92  2.5.sup.d -40.0.sup.e                                                                >40.0                                     9-NO.sub.2 -10-OH-20(S).sup.f                                                            131 (20.0)                                                                           0    -1.12 2.5.sup.d - 40.0.sup.e                                                               >40.0                                     9-NHAc-10-OH-20(S).sup.f                                                                 220 (40.0)                                                                           0    5.50  2.5.sup.d -40.0.sup.e                                                                >40.0                                     __________________________________________________________________________     .sup.a Treatment schedule Q04DX2; % T/C = survival time of treated/contro     animals × 100; IP using Klucel emulsifier.                              .sup.b Log.sub.10 of initial tumor cell population minus log.sub.10 of        tumor cell population at end of treatment.                                    .sup.c 20(S)Camptothecin (1) and 10hydroxy-20(S)-camptothecin (2) were        used as reference standards: for 1, % T/C (8.0 mg/kg) = 197; for 2, % T/C     (24.0 mg/kg) = 230.                                                           .sup.d Lowest dose administered.                                              .sup.e Highest dose administered.                                             .sup.f Compounds 1 and 2 were used as reference standards: for 1, % T/C       (10.0 mg/kg) = 267; for 2, % T/C (20.0 mg/kg) = 348.                     

The table below shows the topoisomerase I inhibitory activity ofcamptothecin and camptothecin analogs. The cleavable complex assay wasperformed according to the method described by Jaxel et al, CancerResearch, 1989, Vol. 49, pp 1465-1469.

    ______________________________________                                        Cleavable Complex Assay of Camptothecin and Analogs                                                         EC.sub.50 **                                    Compound                                                                              Name*                 μg/mL                                        ______________________________________                                        1       10,11-MDO-20(S)-CPT   ˜.01 μg/mL                             2       10,11-MDO-20(RS)-CPT  ˜.02 μg/mL                             3       10,11-MDO-20(S)-CPT, Na.sup.+  SALT                                                                 ˜0.1 μg/mL                             4       9-AMINO-20(RS)-CPT    ˜0.2 μg/mL                             5       10,11-MDO-20(RS)-CPT, Na.sup.+  SALT                                                                ˜0.2 μg/mL                             6       20(S)-CPT             ˜0.2 μg/mL                             7       20(RS)-CPT            ˜0.8 μg/mL                             8       20(RS)-CPT, Na.sup.+  SALT                                                                          ˜0.9 μg/mL                             9       9,10-MDO-20(RS)-CPT   ˜2 μg/mL                               10      9-AMINO-20(RS)-CPT, Na.sup.+  SALT                                                                  ˜10 μg/mL                              11      20(R)-CPT             >10 μg/mL                                    ______________________________________                                         *Abbreviations                                                                CPT = Camptothecin                                                            MDO = Methylenedioxy                                                          **EC.sub.50 is the concentration of a compound which gives 50%                topoisomerase I inhibition as revealed by cleavable complex formation. Al     EC.sub.50 values represent the mean of several independent assays; all        values are normalized with respect to #6, 20(S)CPT, which was always          assayed as a control.                                                    

The cleavable complex assay correlates well with in vivo anti-tumoractivity in animal models for camptothecin analogs. See Hsiang et al,Cancer Research, 1989, 49:4385-4389 and Jaxel et al, Cancer Research,1989, 49:1465-1469.

                                      TABLE III                                   __________________________________________________________________________    Comparative Activities of Ring A Modified and Homologated                     Camptothecin Analogues in Mouse Leukemia Assays.sup.a,b                       Camptothecin                                                                             max % T/C                                                                            no. cures                                                                          K.sub.E.sup.c at max                                                                active dose                                                                          toxic dose,                               derivative (dose, mg/kg)                                                                        out of 6                                                                           % T/C range, mg/kg                                                                         mg/kg                                     __________________________________________________________________________    10-aza-20(RS).sup.a,d                                                                    162 (2.5)                                                                            0    1.61  1.25.sup.e -2.5                                                                      5.0                                       12-aza-20(RS).sup.b,f                                                                    175 (32.0)                                                                           0           .sup. 8.0-32.0                                                                      >32.0                                     A-nor-9-thia-20(RS).sup.a,d                                                              193 (25.0)                                                                           0    -0.94 3.12.sup.e -25.0                                                                     50.0.sup.g                                __________________________________________________________________________     .sup.a Denotes testing in L1210 system; treatment schedule Q04DX2; % T/C      survival time of treated/control animals × 100; IP using Klucel         emulsifier.                                                                   .sup.b Denotes testing in P388 system; treatment schedule Q04DX3; % T/C =     survival time of treated/control animals × 100; IP using Klucel         emulsifier.                                                                   .sup.c Log.sub.10 of initial tumor cell population minus log.sub.10 of        tumor cell population at end of treatment.                                    .sup.d 20(S)Camptothecin sodium was used as a reference standard: % T/C       (40.0 mg/kg) = 215.                                                           .sup.e Lowest dose administered.                                               .sup.f 20(S)Camptothecin was used as a reference standard: % T/C (4.0        mg/kg) = 197.                                                                 .sup.g Highest dose administered.                                        

Pharmaceutical compositions containing the novel camptothecin analogsare also within the scope of the present invention. These pharmaceuticalcompositions may contain any quantity of a camptothecin analog which iseffective to inhibit topoisomerase I in vitro or in vivo or exhibitanti-leukemic activity in vivo. Mammals such as humans are treatablewith the inventive compositions. Typical in vivo doses within the scopeof the invention are from 0.1-60 mg of camptothecin analog per kg ofbody weight. A particularly preferred range is 1-40 mg/kg.

There may also be included as part of the composition pharmaceuticallycompatible binding agents, and/or adjuvant materials. The activematerials can also be mixed with other active materials which do notimpair the desired action and/or supplement the desired action. Theactive materials according to the present invention can be administeredby any route, for example, orally, parenterally, intravenously,intradermally, subcutaneously, or topically, in liquid or solid form.

A preferred mode of administration of the compounds of this invention isoral. Oral compositions will generally include an inert diluent or anedible carrier. They may be enclosed in gelatin capsules or compressedinto tablets. For the purpose of oral therapeutic administration, theaforesaid compounds may be incorporated with excipients and used in theform of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like. These preparations should contain atleast 0.1% of active compound but may be varied depending upon theparticular form.

The tablets, pills, capsules, troches and the like may contain thefollowing ingredients: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, corn starch and thelike; a lubricant such as magnesium stearate or Sterotes; a glidant suchas colloidal silicon dioxide; and a sweetening agent such as sucrose orsaccharin or flavoring agent such as peppermint, methyl salicylate, ororange flavoring may be added. When the dosage unit form is a capsule,it may contain, in addition to material of the above type, a liquidcarrier such as a fatty oil. Other dosage unit forms may contain othervarious materials which modify the physical form of the dosage unit, forexample, as coatings. Thus tablets or pills may be coated with sugar,shellac, or other enteric coating agents. A syrup may contain, inaddition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors. Materials used inpreparing these various compositions should be pharmaceutically pure andnon-toxic in the amounts used.

For the purposes of parenteral therapeutic administration, the activeingredient may be incorporated into a solution or suspension.

The solutions or suspensions may also include the following components:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

The dosage values will vary with the specific severity of the diseasecondition to be alleviated. Good results are achieved when the compoundsdescribed herein are administered to a subject requiring such treatmentas an effective oral, parenteral or intravenous dose. It is to beunderstood that for any particular subject, specific dosage regimensshould be adjusted to the individual need and the professional judgmentof the person administering or supervising the administration of theaforesaid compound. It is to be further understood that the dosages setforth herein are exemplary only and they do not limit the scope orpractice of the invention. The dosages may be administered at once, ormay be divided into a number of smaller doses to be administered atvarying intervals of time.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES Synthesis of Tricyclic Compound 116-Cyano-7-methyl-1,5-dioxo-Δ⁶(8) -tetrahydroindolizine (Compound 3).

Ethyl acetopyruvate was prepared from acetone and diethyl oxalate asdescribed in Org. Synthesis, Coll. Vol. 1, 238 (1958). Further reactionwith triethylorthoformate and ammonium chloride in ethanol afforded theknown enol ether 1. See L. Claisen, Chem. Ber., 40, 3903 (1907).

Ethyl(2-ethoxy-4-oxo)-pent-2-enoate(1) (100.01 g, 0.538 mol) was addedgradually to a preheated (45°), mechanically stirred mixture of K₂ CO₃(79.04 g, 0.573 mol) and cyanoacetamide (48.46 g, 0.577 mol) in DMF(960) mL). The mixture was kept at 45° for 18 h, whereupon the thick,red slurry was treated dropwise with freshly distilled methyl acrylate(360 mL, 343 g, 3.99 mol). After 72 hr at 45°, the red suspension wasfiltered, dissolved in 5 liters of water, and acidified to pH 1.5 withconcentrated HC1. Crude bicyclic ester 2 (127.98 g) was collected byfiltration as a pink solid. Without further treatment, 2 was refluxed ina solution concentrated of HC1 (800 ml) and glacial HOAc (800 ml) for 2hr. Removal of the solvents in vacuo gave the bicyclic pyridone 3 (39.66g, 39% based on 1).

6-Cyano-1,1-(ethylenedioxy)-7-methyl-5-oxo-Δ⁶(8) -tetrahydroindolizine(Compound 4)

Compound 3 (10.54 g, 0.056 mol) as a stirred solution in CH₂ Cl₂ (500ml) was treated at room temperature under N₂ with ethylene glycol (6.85ml, 7.63 g, 0.123 mol) and Me₃ SiCl (31.30 ml, 26.89 g, 0.247 mol) andleft at ambient temperature (20°) for 65 hr. The solution was filteredto remove some black suspended material before washing with 1 M aq NaOHsolution. The organic phase was washed with brine, filtered throughCelite and evaporated to afford ethylene ketal 4 (10.26 g, 79%) as apink solid.

6-Cyano-1,1-(ethylenedioxy)-7-[(ethoxycarbonyl)methyl]-5-oxo-Δ⁶(8)-tetrahydroindolizine (Compound 5)

The ketal 4 (5.0 g, 0.022 mol) was refluxed in a suspension of KH (11.9g, 0.068 mol) in toluene (40 mL) for 10 min. Diethyl carbonate (6.79 g,0.058 mol) and a catalytic amount (0.31 g, 6.7 mmol) of absolute ethanolwere added and refluxing continued for 3 hr. The dark solid was crushedand the resulting suspended salt of 5 was collected by filtration. Thesalt was neutralized by the careful addition of cold aqueous HOAc. Waterwas added and the product extracted into CH₂ Cl₂. Following a wash withbrine and drying (Na₂ SO₄), evaporation of the CH₂ Cl₂ afforded crude 5.Purification by silica gel chromatography (2% MeOH in CHCl₃) andrecrystallization (MeOH) gave pure 5 (4.97 g, 76%).

6-Cyano-1,1-(ethylenedioxy)-7-[1'(ethoxycarbonyl)-propyl]-5-oxo-Δ.sup.6(8)-tetrahydroindolizine (Compound 6)

A stirred solution of the ester 5 (4.01 g, 0.0132 mol) in anhydrous DME(70 mL) at -78° C. was treated with potassium tert-butoxide (1.7 g, 15mmol). After 5 min, EtI (8.24 g, 0.053 mol) was added over a 5 minperiod. After stirring for 1.5 hr at -78° C., the mixture was left towarm to room temperature overnight. Water was added and the productextracted into CH₂ Cl₂. After washing with brine and drying (Na₂ SO₄),CH₂ Cl₂ was evaporated to give the ester 6 (4.3 g, 98%).

6-(Acetamidomethyl)-1,1-(ethylenedioxy)-7-[1'-(ethoxycarbonyl)propyl]-5-oxo-Δ⁶(8)-tetrahydro indolizine (Compound 7)

A solution of the ester ketal 6 (2.0 g, 6.0 mmol) in acetic anhydride(30 mL) and HOAc (10 mL) was hydrogenated for 6 hr at 45° C under 50 psiin the presence of Raney nickel (3 g; washed with HOAc). The catalystwas removed by filtration and the solvent removed in vacuo to give 7(2.3 g, 100%) as an oil. Purification by silica gel columnchromatography (2% MeOH in CHCl₃) gave pure 7 as an oil.

6-(Acetoxymethyl)-1,1-(ethylenedioxy)-7-[1'-(ethoxycarbonyl)propyl]-5-oxo-.DELTA.⁶(8)-tetrahydroin dolizine (Compound 9)

A cooled solution of amide 7 (2.3 g, 6.0 mmol) in Ac₂ O (30 mL) and HOAc(10 mL) was treated with NaNO₂ (1.8 g, 26 mmol) and the reaction mixturestirred for 2 h at 0° C. Inorganic salts were removed by filtration andthe solvent removed in vacuo at room temperature to afford the N-nitrosointermediate 8 as an oil. Compound 8 was converted directly to the titleacetoxy compound 9 by refluxing overnight in CCl₄. The solution waswashed with water, dried (Na₂ SO₄) and the solvent removed in vacuo togive 9 (2.3 g 100%) as an oil.

1,1'Ethylenedioxy-5-oxo-(5'-ethyl-5'-hydroxy-2'H,5'H,6'H-6-oxopyrano)-[3',4'-f]-Δ⁶,8-tetrahydroindolizine (Compound 10)

Oxygen was bubbled through a mixture of6-(acetoxymethyl)-1,1-(ethylenedioxy)-7-[1'-ethoxycarbonyl)-propyl]-5-oxo-Δ⁶,8-tetrahydroindoliz ine (Compound 9, 405 mg, 1.07 mmol), anhydrous K₂ CO₃(148 mg 1.07 mmol) and methanol (7.5 mL) for 24 hr. The solution wascooled in an ice bath and made acidic (pH 2-4) by addition of 1N H₂ SO₄.Most of the methanol was removed in vacuo at room temperature, and water(20 mL) was added. The aqueous solution was extracted with CH₂ Cl₂ (3×20mL), dried (NaSO₄) and evaporated to give a solid which was crystallizedfrom CH₂ Cl₂ -hexane to give 280 mg (85%) of 10: mp 179°-181° C.;ν_(max) (CHCl₂) 1740, 1660 cm⁻¹ ; ¹ H-NMR (CDC1₃) δ 0.91 (t, 3, J=7 Hz,CH₂ CH₃), 1.75 (q, 2, J=7 Hz, CH₂ CH₃), 2.35 (t, 2, J=6.5 Hz, CH₂ α toketal), 4.1 (m, 6, OCH₂ CH₂ O and CH₂ N), 5.30 (m, 2, ArCH₂ O), 6.87 (s,1, pyridone). Anal. Calcd for C₁₅ H₁₇ NO₆ : C, 58.63; H, 5.54; N, 4.56.Found: C, 58.72, H, 5.68; N, 4.57.

5'RS-1,5-Dioxo-(5'-ethyl-5'-hydroxy-2'H,5'H,6'H-6-oxopyrano)-[3',4',f]-Δ⁶,8 -tetrahydroindolizine (Compound 11)

A solution of 10 (3.88 g, 12.6 mmol) in 2N H₂ SO₄ (50 mL) and DME (50mL) was heated for 24 hr under N₂. The reaction mixture was concentratedto one half its volume in vacuo, diluted with H₂ O (100 mL) andextracted with CH₂ Cl₂ (5×50 mL). The organic layer was dried (Na₂ SO₄)and evaporated to yield a solid which was crystallized from CH₂ Cl₂-hexane to yield 2.68 g (80%) of 11 as a light brown solid: mp 185°-187°C.; ν_(max) (CHCl₃) 1750 (shoulder, ketone), 1745 (lactone), 1660 cm⁻¹(pyridone); ¹ H-NMR (CDCl₃) δ 0.91 (t, 3, J=7 Hz, CH₂ CH₃), 1.80 (q, 2,J=7 Hz, CH₂ CH₃), 2.93 (t, 2, J=6.5 Hz, CH₂ C=0), 4.30 (t, 2, J=6.5 Hz,CH₂ N), 5.35 (m, 2, ArCH₂ O), 7.17 (s, 1, aromatic H). Anal Calcd forC₁₃ H₁₃ NO₅ : C, 59.32; H, 4.94; N, 5.32. Found: C, 59.12, H, 4.91; N,5.16.

Synthesis of Camptothecin Analogs Synthesis of11-hydroxy-20(RS)-camptothecin

11-hydroxy-20(RS)-camptothecin is prepared from11-methoxy-20(RS)-camptothecin by demethylation of the latter withhydrobromic acid as follows:

11-Methoxy-20(RS)-camptothecin

A mixture of 4-methoxy-2-aminobenzaldehyde (180 mg, 1.19 mmol) and thetricyclic ketone 11 (300 mg, 1.14 mmol) in toluene (18 mL) was heatedunder N₂ in a flask equipped with a Dean-Stark trap. At refluxp-toluenesulfonic acid (5 mg) was added, and the red-brown solution washeated for an additional 2 hr. The toluene was removed under reducedpressure to give a brown solid which was treated with water (10 mL) andchloroform (20 mL). The aqueous phase was extracted with additionalchloroform (3×20 mL) and the combined extracts dried (Na₂ SO₄).Evaporation gave a brown solid which was recrystallized frommethanol-chloroform to give 216 mg (50%) of compound as a tan solid:275°-279° C.; mass spectrum (electron impact), m/z 378.1219 M⁺ ; C₂₁ H₁₈N₂ O₅ requires 378.1214; νmax (KBr) 3480 (OH), 1745 (lactone), 1660(pyridone), 1622, 1236 and 1152 cm⁻¹ ; ¹ H-NMR (DMSO-d₆) δ 0.87 (t, 3,J=7 Hz, H-18), 1.85 (m, 2, H-19), 3.95 (s, 3, 11-OCH₃), 5.24 (s, 2,H-5), 5.42 (s, 2, H-17), 7.32 (s, 1, H-14), 7.37 (dd, 1, J=9, 2.5 Hz,H-10), 7.56 (d, 1, J=2.5 Hz, H-12), 8.02 (d, 1, J=9 Hz, H-9), 8.60 (s,1, H-7).

11-Hydroxy-20(RS)-camptothecin

11-methoxy-20(RS)-camptothecin (75 mg) was combined with 48% aqueous HBr(2.5 mL) and heated at reflux for 6 hr. The red-brown mixture wasstripped of solvent under high vacuum. Chromatography of the residuethrough silica gel (15 g) (7% MeOH--CHCl₃) gave the 11-hydroxy compound(33 mg, 45%) which was further purified by recrystallization from 13%MeOH in CHCl₃ : mp 323°-326° C.; mass spectrum (electron impact), m/z364.1054 M⁺, C₂₀ H₁₆ N₂ O₅ requires 364.1059; ν_(max) (KBr) 3450, 1742,1654, 1613, 1592, 1570, 1245 cm⁻¹ ; λ_(max) (EtOH), 224 (log ε 4.58),259, (4.39), 353 (4.16), 371 (4.19), 387 (4.20); ¹ H-NMR (DMSO-d₆): δ0.88 (t, 3, J=7 Hz, H-18), 1.85 (m, 2, H-19), 5.20 (s, 2, H-5), 5.41 (s,2, H-17), 6.51 (br s, 1, OH-20), 7.26 (dd, 1, J=9, 2.5 Hz, H-10), 7.28(s, 1, H-14).

10-Hydroxy-20(RS)-camptothecin

This compound is prepared in a manner analogous to that described forthe 11-hydroxycamptothecin using e5-methoxy-2-aminobenzaldehyde which isreacted with the tricyclic ketone 11 in the presence ofp-toluenesulfonic acid. The product is 10-methoxy20(RS)-camptothecinwhich on treatment with refluxing hydrobromic acid as described for11-hydroxycamptothecin, gives 10-hydroxy-20(RS)-camptothecin.

9-Methoxy-20(RS)-camptothecin and 9-Hydroxy-20(RS)-camptothecin

In a manner analogous to that described for11-methoxy-20(RS)-camptothecin, 6-methoxy-2-aminobenzaldehyde is treatedwith the tricyclic 11 ketone in the presence of p-toluenesulfonic acidyielding 9.methoxy-20(RS)-camptothecin. Demethylation with hydrobromicacid gives 9-hydroxy-20(RS)-camptothecin.

10-Nitro-20(RS)-camptothecin

A mixture of 2-amiho-5-nitrobenzaldehyde (95 mg, 0.57 mmol) and thetricyclic ketone 11 (150 mg, 0.57 mmol) was heated at 120° C. for 10min. The temperature was raised to 160° C., and the dark molten mass waskept at this temperature for 1.5 hr with occasional stirring.Chromatography of the residue through silica gel (20 g) using 0.5% MeOHin CHCl₃ afforded the title compound (108 mg) as a yellow solid; mp297°-300° C. (decomp.); mass spectrum (electron impact), m/z 393.0965M⁺, C₂₀ H₁₅ N₃ O₆ requires 393.0960; ν_(max) (KBr) 3450 (OH), 1745(lactone), 1660 (pyridone), 1620, 1350, and 1160 cm⁻¹ ; ¹ H-NMR (TFA-d₁)δ 1.14 (t, 3, J=7 Hz, H-18), 2.15 (m, 2, H-19), 5.88 (s, 2, H-5), 5.68(Abq, 2, J=17 Hz, Δ.sub.γ =85 Hz, H-17), 8.43 (s, 1, H-14), 8.70 (d, 2,J=8 Hz, H-12), 9.05 (d, 2, J=8 Hz, H-11), 9.35 (s, 1, H-9), 9.60 (s, 1,H-7).

10-Amino-20(RS)-camptothecin

A suspension of 10-nitro-20(RS)-camptothecin (100 mg) and 10% Pd/C (40mg) in absolute EtOH (40 mL) was stirred in an atmosphere of H₂ at roomtemperature for 30 min. Filtration through Celite and removal of thesolvent under reduced pressure gave a tan yellow solid (86 mg crude).Recrystallization from 13% MeOH/CHCl₃ gave the pure product (30 mg) asan olive-yellow solid: mp, softening at 135° C., gradual blackening uponfurther heating; mass spectrum (electron impact), m/z 363.116 M⁺ ; C₂₀H₁₇ N₃ O₄ requires 363.1218; ν_(max) (KBr) 3440 (OH, NH₂), 1750(lactone), 1660 (pyridone) cm⁻¹ ; ¹ H-NMR (TFA-d) δ 1.06 (t, 3, J=7Hz,H-18), 2.08 (d, J=7Hz, H-17), 5.89 (s, 2, H-5), 5.70 (Abq, 2, J=17Hz,Δ.sub.γ =85Hz, H-17), 8.34 (d, J =9Hz, H-12), 8.64 (d, J= 9Hz, H-11),9.26 (s, 1, H-(), 9.43 (s, 1, H-7).

9-Nitro-20(RS)-camptothecin and 9-Amino-20(RS)-camptothecin

A mixture of 2-amino-6-nitrobenzaldehyde is treated with the tricyclicketone 11 in the manner described for the 10-nitro series above yielding9-nitro-20(RS)-camptothecin. This compound, after reduction withpalladium/carbon, yielded 9-amino20(RS)-camptothecin. Alternatively, the9-amino compound is obtained in one step by reaction of2,6-diaminobenzaldehyde with ketone 11.

11-Nitro-20(RS)-camptothecin and 11-Amino-20(RS)-camptothecin

In a manner similar to that described for 10-nitro-20(RS)-camptothecin,a mixture of 2-amino-4-nitrobenzaldehyde is treated with the tricyclicketone 11 yielding 11-nitro-20(RS)-camptothecin which in turn is reducedto 11-amino-20(RS)-camptothecin by palladium/carbon. Alternatively, the11-amino20(RS)-camptothecin is obtained by reaction of2,4-diaminobenzaldehyde with ketone 11.

10,11-Dihydroxy-20(RS)-camptothecin

A solution of the crude dibenzyloxy aminoacetal (400 mg) and thetricyclic ketone 11 (132 mg, 0.5 mmol) in toluene (60 mL) was refluxedfor 8 hr. It was filtered hot, and the pure dibenzylether was collectedupon cooling (200 mg, 81%); mp 276° C. ν_(max) (KBr) 3440, 1740, 1650,1590, 1490, 1440, 1380, 1250, 1140, 1100 cm⁻¹ ; 250 MHz ¹ H-NMR(DMSO-d₆) δ 0.88 (t, 3, J=7 Hz, H-18), 1.86 (m, 2, H-19), 5.22 (s, 2,H-17), 5.34 (s, 2, 10--OCH₂ --C₆ H₅), 5.39 (s, 2, 11--OCH₂ --C₆ H₅),5.41 (s, 2, H-5), 6.5 (s, 1, OH), 7.25 (s, 1, H-14) 7.35-7.65 (m, 12,H-9, 12, --OCH₂ --C₆ H₅), 8.44 (s, 1, H-7). Anal. calcd for C₃₄ H₂₈ N₂O₆ : C, 72.84; H, 5.03; N, 5.00. Found C, 72.91; H, 5.09; N, 4.96.

The dibenzyl ether (130 mg, 0.23 mmol) was mildly refluxed for 2 hr in24% HBr (50 mL). The acid was removed, and the residue was dissolved inhot methanol (50 mL). Ether (50 mL) was added at room temperature andthe yellow powdery dihydroxy camptothecin hydrobromide was collected(122 mg, 77%) mp > 300° C. ν_(max) (KBr) 3400 (b), 1740, 1655, 1585,1545, 1510, 1395, 1300, 1270, 1200, 1160 cm⁻¹ ; ¹ H NMR (DMSO, d₆): δ0.88 (t, 3, J=7 Hz, H-18), 1.85 (m, 2, H-19), 5.20 (s, 2, H-17), 5.42(s, 2, H-5), 7.31 (s, 2, H-9, H-14), 7.40 (s, 1, H-12), 8.45 (s, 1,H-7). Anal. calcd for C₂₀ H₁₇ BrN₂ O₆ · 0.5 H₂ O: C, 51.08; H, 3.86; N,5.95; Br, 16.99. Found C, 51.09; H, 4.04; N, 5.78; Br, 16.83.

Dihydroxy hydrobromide salt (110 mg, 0.23 mmol) was suspended in water(10 mL). Sodium hydroxide (0.1 N, 7.2 mL) was added and the mixture wasagitated. The resulting clear solution was acidified using 5N HC1; andafter an hour, the sample was centrifuged, the supernatant liquid wasdecanted and the process repeated with additional water (20 mL). Theresidue was dried (78 mg, 74%); mp > 300° C. ν_(max) (KBr): 3490, 3000(b), 1740, 1645, 1590, 1460, 1385, 1265, 1190, 1150 cm⁻¹. ¹ H NMR (DMSO,d₆): δ 0.88 (t, 3, J=7 Hz, H-18), 1.87 (q, 2, H-19), 5.20 (s, 2, H-17),5.42 (s, 2, H-5), 7.35 (s, 1, H-14), 7.44 (s, 1 H-9), 7.52 (s, 1, H-12),8.51 (s, 1, H-7). Anal. calcd for C₂₀ H₁₆ N₂ O₆ · 0.75 H₂ O: C, 61.06;H, 4.44; N, 7.12. Found C, 61.12; H, 4.44; N, 7.09.

10-Chloro-20(RS)-camptothecin

This compound was prepared by treating 5-chloro-2-aminobenzaldehyde withthe tricyclic ketone 11.

A solution of the 5-chloro-2-aminobenzaldehyde (80 mg, 0.51 mmol) andthe tricyclic ketone 11 (100 mg, 0.38 mmol) in toluene (60 mL) wasrefluxed for 15 min. p-Toluenesulfonic acid (10 mg) was then added, andrefluxing was continued for an additonal 5 hr. The solvent was removedin vacuo and the residue chromatographed (silica gel 60, 2% MeOH-CHCl₃).The product obtained was recrystallized from CHCl₃ -MeOH-EtOAc; mp 270°C., 60 mg (41%). ν_(max) (KBr), 3430, 1745, 1655, 1600, 1495, 1230, 1160cm⁻¹. 250 MHz ¹ H-NMR (TFA-d₁) δ 1.15 (t, 3, J=7 Hz, H-18), 2.16 (m, 2,H-19), 5.73 (ABq, 2, J=17 Hz, Δ.sub.γ =85 Hz, H-17), 5.84 (s, 2, H-5),8.29 (d, 1, J=9 Hz, H-11), 8.35 (s, 1, H-14), 8.40 (s, 1, H-9), 8.45 (d,1, J=9 Hz, H-12), 9.31 (s, 1, H-7). Anal. calcd for C₂₀ H₁₅ ClN₂ O₄ 0.5H2O: C, 61.47; H, 4.12; N, 7.17; Cl, 9.07. Found C, 61.41; H, 4.12; N,7.12; Cl, 9.11.

10-Methyl-20(RS)-camptothecin

5-Methyl-2-aminobenzaldehyde was treated with the tricyclic ketone 11 togive the title compound.

The tricyclic ketone 11 (130 mg, 0.5 mmol) and the5-methyl-2-aminobenzaldehyde (560 mg) in toluene (60 mL) were refluxedfor 0.5 hr. Acetic acid (1 mL) and p-toluenesulfonic acid (35 mg) wereadded, and refluxing was continued for an additional 5 hr. The solventwas removed in vacuo, and the residue was triturated with warm ether (30mL). The product was recrystallized from chloroform-methanol-ether toyield pure compound (102 mg, 57%), mp 278°-280° C. (KBr) 3460, 2980,1740, 1655, 1590, 1550, 1470, 1450, 1370, 1260, 1240, 1160, 1050 cm⁻¹.250 MHz ¹ H-NMR (DMSO-d₆) δ 0.89 (t, 3, J=7 Hz, H-18), 1.87 (q, 2,H-19), 2.54 (s, 3, 10-CH₃), 5.24 (s, 2, H-17), 5.42 (s, 1, H-5), 7.31(s, 1, H-14), 7.69 (d, 1, J=8.6 Hz, H-11), 7.86 (s, 1, H-9), 8.05 (d, 1,J=8.6 Hz; H-12), 8.55 (s, 1, H-7). Anal. calcd for C₂₁ H₁₈ N₂ O₄ · 0.25H₂ O: C, 68.75; H, 5.08; N, 7.64. Found C, 68.74; H, 5.08; N, 7.64.

11-Formyl-20(RS)-camptothecin

2-Nitroterephthaldicarboxaldehyde was converted to the ethylene diacetalby conventional methods and reduced using Na₂ S. A solution of the nitrodiacetal (4.1 g, 17.5 mmol), Na₂ S (14 g) in 80% ethanol (15 mL) wasrefluxed for 1 hr. Ethanol was removed in vacuo, the reaction mixturewas diluted with water (10 mL) and the aqueous phase was extracted withCH₂ Cl₂ (4 · 50 mL). The organic phase was washed with water, dried(MgSO₄), and evaporated to give the aminodiacetal, which wasrecrystallized from ethyl acetatehexane (2.8 g, 78%); mp 76° C. ν_(max)(KBr) 3480, 3395, 3000, 2960, 2900, 1625, 1445, 1395, 1085, 950 cm⁻¹. 60MHz ¹ H NMR (CDCl₃ -D₂ O) δ 4.0 (m, 8, --OCH₂ CH₂ O--), 5.6 (s, 1,--O--CH--, C-4), 5.7 (s, 1, --O--CH--, C-1), 6.6 (s, 1, H-3), 6.65 (d,1, J=8 Hz, C-5), 7.2 (d, 1, J=8 Hz, H-6). Anal. calcd for C₁₂ H₁₅ NO₄ :C, 60.66; H, 6.36; N, 5.90. Found C, 60.79; H, 6.41; N, 5.84.

A solution of the tricyclic ketone 11 (265 mg, 1.0 mmol), aminodiacetal(500 mg, 2.1 mmol), 300 mg initally, 100 mg each at intervals of 5 and10 hr) in toluene (70 mL) was refluxed for 0.5 hr. Acetic acid (2 mL)was added and refluxing continued for 18 hr. The solvent was evaporatedin vacuo, and the residue was taken up in 75% methanol (250 mL). Conc.HC1 (3 mL) was added and the reaction mixture heated at 50°-60° C. for24 hr. The mixture was filtered, and the residue was washed with waterand recrystallized from CHCl₃ --MeOH--EtOAc. mp: 276°-279° C. (175 mg,45%). ν_(max) (KBr) 3460, 1745, 1690, 1655, 1600, 1200, 1150, 1135 cm⁻¹.250 MHz ¹ H NMR (TFA-d₁), δ 1.16 (t, 3, J= 7 Hz, H-18) 2.16 (q, 2, J=7Hz, H-19), 5.78 (ABq, 2, J=18 Hz, Δ.sub.γ =85 Hz, H-17), 5.89 (s, 2,H-5), 8.43 (s, 1, H-14), 8.66 (d, 1, J=8.5 Hz, H-10), 8.60 (d, 1, J=8.5Hz, H-9), 9.12 (s, 1, H-12), 9.49 (s, 1, H-7), 10.42 (s, 1, CHO). Anal.calcd. for C₂₁ H₁₆ N₂ O₅ · H₂ O: C, 64.01; H, 4.56; N, 7.11. Found C,64.08, H, 4.21; N, 6.84.

11-Cyano-20(RS)-camptothecin

A mixture of 11-formyl-20(RS)-camptothecin (225 mg, 0.6 mmol),hydroxylamine hydrochloride (50 mg, 0.72 mmol), sodium formate (90 mg,1.3 mmol), and formic acid (6 mL) was refluxed for 1.5 hr. The mixturewas evaporated to dryness in vacuo, and the residue was washed withwater, dried and chromatographed (silica gel 60, 0.5% MeOH-CHCl₃) andrecrystallized from CHCl₃ -EtOAc to yield the 11-cyano compound (65 mg,29%): mp 288° C. ν_(max) (KBr) 3400, 2235, 1735, 1655, 1590, 1450, 1400,1230, 1150, 1110, 1045 cm⁻¹. 250 MHz ¹ H NMR (DMSO-d₆): δ 0.88 (t, 3,J=7 Hz, H-18), 1.88 (m, 2, H-19), 5.32 (s, 2, H-17), 5.44 (s, 2, H-5),7.37 (s, 1, H-14), 7.98 (d, 1, J=8.5 Hz, H-10), 8.32 (d, 1, J=8.5 Hz,H-9), 8.74 (s, 1, H-12), 8.80 (s, 1, H-7). Anal. calcd for C₂₁ H₁₅ N₃ O₄· 1.5 H₂ O: C, 62.99; H, 4.52; N, 10.49. Found C, 62.99; H, 3.95; N,10.20.

Alternatively, 11-cyano-20(RS)-camptothecin can be prepared by thereaction of 5-cyano-2-aminobenzaldehyde with the tricyclic ketone 11.

Preparation of Camptothecin Analogs with Modified A Ring Structure

The reaction of the tricylic ketone 11 with suitable precursors otherthan substituted ortho-aminobenzaldehydes can be used to give active newcamptothecin analogs exemplified by the following non-limiting examples:

10-Aza-20(RS)-camptothecin

A solution of 4-aminonicotinaldehyde (24.2 mg, 0.198 mmol), thetricyclic ketone 11 (53.5 mg, 0.203 mmol) and p-TsOH · H₂ O (2 mg) intoluene (25 mL) was refluxed for 4 days using Dean-Stark trap. Thesolvent was removed under reduced pressure, and the residue waschromatographed through silica gel (20 g) using CHCl₃ -acetone-MeOH(5:1:1). The product was crystallized from 13% MeOH in CHCl₃ and EtOAc:mp 289°-292° C.; mass spectrum (electron impact), m/z 349.1061 M⁺ ; C₁₉H₁₅ N₃ O₄ requires 349.1066; ν_(max) (KBr) 3320 (OH), 1730 (lactone),1650 (pyridone), 1600 (aromatic) cm⁻¹ ; ¹ H NMR (CDCl₃) 1.05 (t, 3,J=7.3 Hz, H-18), 1.92 (m, 2, H-19), 5.35 (s, 2, H-5), 5.52 (ABq, 2, J=18Hz, Δ.sub.γ =85 Hz, H-17), 7.74 (s, 1, H-14), 8.04 (d, 1, J=5.5 Hz,H-12), 8.53 (s, 1, H-7), 8.84 (d, J=5.5 Hz, H-11), 9.4 (s, 1, H-9).

A-Nor-9-thia-20(RS)-camptothecin

This sulfur containing camptothecin analog is prepared by the reactionof 3-amino-2-formylthiophene with tricyclic ketone 11.

A solution of 3-amino-2-formylthiophene (79 mg, 0.62 mmol) and thetricyclic ketone 11 (96 mg, 0.37 mmol) in toluene (1.5 mL) was broughtto reflux and then cooled before adding a crystal of p-toluenesulfonicacid. The mixture was refluxed for 2.5 hr under N₂, cooled and theprecipitate filtered. The crude material was chromatographed on silicagel (20 g) by elution with 2% MeOH in CHCl₃. Crystallization of theproduct from 13% MeOH-CHCl₃ and EtOAc yielded the title compound as ayellow solid (19 mg, 15%): mp 297°-298° C.; ν_(max) 1740 (lactone), 1655cm⁻¹ (pyridone); ¹ H NMR (TFA-d₁) δ 1.05 (t, 3, J=7 Hz, H-18), 2.07 (q,2, J=7 Hz, H-19), 5.60 (m, 2, H-17), 5.65 (s, 2, H-5), 7.89 (d, J=6 Hz,H-11), 8.05 (s, 1, H-14), 8.57 (d, J=6 Hz, H-10), 9.23 (s, 1, H-7).Anal. (C₁₈ H₁₄ N₂ O₄ S), calcd. C, 61.02; H, 3.95; N, 7.91. Found C,60.65; H, 4.01; N, 7.78.

10,11-Methylenedioxy-20(RS)-camptothecin

The required ortho-aminoaldehyde was prepared by reduction of2-nitropiperonal. This compound (60 mg, 0.36 mmol) and the tricyclicketone 11 (53 mg, 0.20 mmol) were refluxed for 8 hr in toluene (30 mL)containing p-TsOH . H₂ O (8 mg). The solvent was removed in vacuo, thered residue adsorbed onto Celite (1 g) and chromatographed throughsilica gel (10 g) using 3% MeOH in CHCl₃. Concentration of theappropriate fractions gave 10,11-methylenedioxy-20(RS)-camptothecin (36mg, 45%) as a pale tan solid. Crystallization of this material fromCHCl₃ gave the analytical sample as a cream-colored solid: mp > 250° C.(decomp); ν_(max) (KBr) 1750 (lactone), 1655 (pyridone), 1585 cm⁻¹(aromatic); ¹ H NMR (TFA-d₁) δ 1.15 (t, 3, J=7 Hz, H-18), 2.16 (q, 2,J=7 Hz, H-19), 5.76 (ABq, 2, J=17 Hz, Δ.sub.γ =85 Hz, H-17), 5.73 (s, 2,H-5), 6.44 (s, 2, OCH₂ O), 7.55 (s, 1, H-14), 7.69 (s, 1, H-9), 8.16 (s,1, H-12), 9.05 (s, 1, H-7). Anal. calcd for C₂₁ H₁₆ N₂ O₆ : 392.1008.Found 392.1009 (C₂₁ H₁₆ N₂ O₆ · 1.0 H₂ O).

10,11-Methylenedioxy-20(RS)-camptothecin Sodium Salt (10,11-MDCPT SodiumSalt)

The title compound was prepared from10,11-methylenedioxy-20(RS)-camptothecin (Wani et al., J. Med. Chem. 29,2358 (1986)) by hydrolytic action of sodium hydroxide. Thus, 10,11-MDCPT(77 mg, 0.194 mmol) was suspended in 90% aqueous methanol (30 mL) andtreated with 0.1 N aqueous sodium hydroxide (1.94 mL, 0.194 mmol). Uponheating at 50°-60° C. for 1 h under nitrogen a clear solution resultedwhich was cooled to ambient temperature and evaporated to dryness. Theresidue was dissolved in distilled water (2 mL) and filtered (0.45micron membrane), and the resulting solution evaporated. The residue wasrecrystallized from ethanol/ether to provide the title compound as apale yellow solid (53 mg, 65%), mp > 300° C.; IR ν_(max) (KBr) 3400(br), 2970, 2920, 1640, 1610, 1560-1580, 1497, 1466, 1370, 1246, 1225,1183, 1030, 1000, 947, 855, 810, 761, 708 and 560-580; ¹ H NMR (DMSO-d₆)δ0.85 (t, 3, J =7 Hz, H-18), 2.09 (m, 2, H-19), 4.74 (ABq, 2, Δ.sub.γ=68 Hz, J=12, 4 Hz, H-17), 5.12 (s, 2, H-5), 5.64 (dd, 1, J =4, 7 Hz,17-OH), 6.17 (s, 1, 20-OH), 7.47 (s, 1, H-14), 7.54 (s, 1, H-9), 7.62(s, 1, H-12, 8.41 (s, 1, H-7).

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method of inhibiting the enzyme topoisomeraseI in a mammal, comprising:administering to said mammal 0.1-60 mg per kgof body weight of a camptothecin selected from the group consisting of10,11-methylenedioxy-20(RS)-camptothecin,10,11-methylenedioxy20(RS)-camptothecin sodium salt and mixturesthereof.
 2. The method of claim 1, wherein said enzyme is humantopoisomerase I.
 3. The method of claim 1, wherein said camptothecin is10,11-methylenedioxy-20(RS)-camptothecin.
 4. The mothod of claim 1,wherein said camptothecin is 10,11-methylenedioxy-20(RS)-camptothecinsodium salt.
 5. A method of inhibiting the enzyme topoisomerase I in amammal, comprising:administering to said mammal 0.1-60 mg per kg of bodyweight of a comptothecin selected from the group consisting of9-amino-20(RS)-camptothecin, 10-amino20(RS)-camptothecin and mixturesthereof.
 6. The method of claim 5, wherein said camptothecin is9-amino-20(RS)-camptothecin.
 7. The method of claim 5, wherein saidcamptothecin is 10-amino-20(RS)-camptothecin.